Abstract

Rho-kinase and protein kinase C (PKC) have each been reported to mediate vasoconstriction via calcium sensitization. However, the relative contributions of these two kinases to vascular contraction, and whether their roles vary between large and small arteries, are largely unknown. We therefore assessed the relative roles of rho-kinase and PKC in mediating vasoconstriction in arteries from three segments of the aortic and mesenteric vasculature. We studied contractile responses of rat isolated thoracic aorta (diameter ≈2 mm), superior mesenteric artery (SMA; ≈1.5 mm), and second order branches of the superior mesenteric artery (BMA; ≈300 μm). The roles of rho-kinase and PKC in mediating contractile responses to phenylephrine, 9,11-dideoxy-9,11-methanoepoxy prostaglandin F2α (U46619), and KCl were assessed by using the rho-kinase inhibitor R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide (Y-27632) (1 and 10 μM) and the PKC inhibitor 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (Ro 31-8220) (5 μM). Contractile responses of aorta and SMA were reduced by either 1 or 10 μM Y-27632 (P < 0.05), whereas responses of BMA were reduced by 10 μM (P < 0.05) but not 1 μM Y-27632. In contrast, Ro 31-8220 partly reduced contractile responses in aorta and SMA (P < 0.05), but it abolished responses of BMA (P < 0.05). Cotreatment with Y-27632 and Ro 31-8220 markedly attenuated contractile responses to phenylephrine and KCl in all vessels, but it had only a moderate inhibitory effect on responses to U46619 in aorta and SMA. Thus, contractile responses of the larger arteries can involve both rho-kinase and PKC to varying degrees. Conversely, contractile responses of small mesenteric resistance arteries seem to be mediated exclusively by PKC, with no apparent role for rho-kinase.

Vascular smooth muscle (VSM) tone is largely dependent on the phosphorylation state of the myosin regulatory light chain (MLC) of myosin II, which is phosphorylated by Ca2+-dependent myosin light chain kinase (MLCK). In its phosphorylated form, MLC interacts with actin filaments, leading to VSM contraction. Conversely, MLC is dephosphorylated by myosin light chain phosphatase (MLCP), which in turn inhibits the actin-myosin II interaction and promotes relaxation. Hence, vascular tone is dependent on the relative activities of MLCK and MLCP, whereby either MLCK activation or MLCP inhibition can increase vascular contractility (Somlyo and Somlyo, 2000; Budzyn et al., 2006).

MLCP inhibition enhances the sensitivity of the VSM contractile apparatus to [Ca2+] and is therefore known as “Ca2+ sensitization”. The small G protein rho (particularly its rhoA isoform) and its downstream effector rho-kinase have been reported to induce VSM contraction via Ca2+ sensitization (Somlyo and Somlyo, 2003; Budzyn et al., 2006). RhoA can be activated after stimulation of heterotrimeric G protein-coupled receptors in response to agonists such as phenylephrine and thrombin (Gong et al., 1997; Seasholtz et al., 1999). In its activated form, rhoA can interact with and activate rho-kinase, which then phosphorylates and inactivates MLCP on its myosin-binding subunit. This leads to an increased proportion of MLC in its phosphorylated state, promoting VSM contraction (Budzyn et al., 2006).

Another mechanism by which Ca2+ sensitization can occur is via activation of protein kinase C (PKC). PKC can be activated physiologically by endogenous diacylglycerol or pharmacologically by phorbol esters (Akopov et al., 1998; Chrissobolis and Sobey, 2002). More recently, it has been demonstrated that pathologically important mediators such as oxyhemoglobin and endothelin-1 can also activate PKC (Wickman et al., 2003; McNair et al., 2004). PKC can mediate Ca2+ sensitization by phosphorylating the phosphoprotein CPI-17 (Kitazawa et al., 2000), yet this phosphorylation has also been reported to occur via rho-kinase (Koyama et al., 2000). Phosphorylated CPI-17 is a potent inhibitor of MLCP, causing physical dissociation of MLCP subunits, rendering it inactive and therefore promoting increased vascular tone.

Although the influence of PKC on vascular tone has been investigated in various vascular beds, studies have thus far yielded conflicting results with regard to the physiological significance of this pathway (Kadokami et al., 1996; Chrissobolis and Sobey, 2001; Woodsome et al., 2001). Furthermore, the relative contributions of rho-kinase and PKC to vascular contraction and whether their roles vary between large and small blood vessels within a vascular bed are not well understood. This study has therefore investigated the relative roles of rho-kinase and PKC in mediating contraction of aorta and small mesenteric arteries and whether there are segmental differences in these roles.

Materials and Methods

All experimental procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee and complied with National Health and Medical Research Council of Australia guidelines. Adult male Sprague-Dawley rats (237-500 g; n = 97) were studied.

After equilibration for 45 min, arterial segments were exposed to an isotonic high K+-containing physiological saline solution (KPSS; [K+]KPSS = 124 mM). KPSS-induced contraction reached a stable level over 10 to 20 min. After washout and return to stable baseline, segments were precontracted to ∼50% of their KPSS response with serotonin (1-10 μM). Sustained relaxation (>70% of precontracted tone) in response to 10 μM acetylcholine confirmed the presence of functional endothelium. In some experiments, endothelium of BMA was removed by gentle rubbing with a human hair. After washout and return to stable baseline, cumulative concentration-response curves were established to the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 mimetic U46619, and the receptor-independent depolarizing agent KCl. Two to three such curves were typically performed per arterial segment. The order in which agonists were applied was randomized and did not affect the responses obtained. All control data were obtained in rings not previously exposed to any inhibitor.

Effects of Rho-Kinase and PKC Inhibition on Vascular Contraction. The effect of the rho-kinase inhibitor Y-27632 (1 or 10 μM) on contractile responses was assessed by treating arterial segments for 30 min before commencing cumulative additions of phenylephrine, U46619, or KCl. Likewise, the effect of the PKC inhibitor Ro 31-8220 (5 μM) on contractile responses was assessed by 30-min treatment before the addition of contractile agents. In some experiments, the effects of combined Y-27632 (1 μM) and Ro 31-8220 (5 μM) treatment on responses to contractile agents were also studied. The effect of rho-kinase or PKC inhibition on contractions to the PKC activator phorbol 12,13-dibutyrate (PdB; 0.1 μM) was also investigated.

Drugs. Y-27632 was obtained from Welfide Corporation (Osaka, Japan). Ro 31-8220 was obtained from Calbiochem (San Diego, CA). All other drugs were obtained from Sigma-Aldrich (St. Louis, MO). Ro 31-8220 was dissolved as a stock solution of 1 mM in 100% dimethyl sulfoxide and diluted in deionized water. All other drugs were dissolved and diluted in deionized water.

Analyses and Statistics. All responses to contractile agents are presented as percentage of the KPSS response of each arterial segment. Each n represents the number of animals used. Statistical analysis was carried out using one-way analysis of variance followed by Dunnett's or Tukey's post hoc tests, as appropriate. P < 0.05 was considered statistically significant.

Effect of Rho-Kinase and PKC Inhibition on Contractile Responses to Phenylephrine. As seen in Fig. 1, a, c, and e, maximum contractile responses to phenylephrine were reduced by 1 μM Y-27632 in aorta and SMA (P < 0.05; Fig. 2, a and b), its inhibitory effect being more pronounced in aorta. Furthermore, Y-27632 had no effect on responses of BMA (Fig. 2c). Ro 31-8220 alone reduced contractile responses of both aorta and SMA (P < 0.05; Fig. 2, a and b), with its inhibitory effect being most marked in SMA. In contrast, responses of BMA were effectively abolished by Ro 31-8220 (P < 0.05; Fig. 2c). Endothelial removal did not alter this effect of Ro 31-8220 in BMA (data not shown). Cotreatment with Y-27632 and Ro 31-8220 virtually abolished contractile responses of all arteries (P < 0.05; Fig. 2, a-c).

Effect of Rho-Kinase and PKC Inhibition on Contractile Responses to KCl. Maximum contractile responses to KCl were modestly attenuated by 1 μM Y-27632 in aorta and SMA (Fig. 3, a and b), whereas Y-27632 did not affect responses of BMA (Fig. 3c). Ro 31-8220 reduced KCl-induced contractile responses of both aorta and SMA (P < 0.05; Fig. 3, a and b), its inhibitory effect being most pronounced in SMA. As seen for phenylephrine, responses of BMA to KCl were abolished by Ro 31-8220 (P < 0.05; Fig. 3c). Furthermore, cotreatment with Y-27632 and Ro 31-8220 profoundly inhibited contractile responses of aorta and effectively abolished responses in SMA and BMA (P < 0.05; Fig. 3, a-c).

Effect of Rho-Kinase and PKC Inhibition on Contractile Responses to U46619. Maximum contractile responses to U46619 were unaffected by 1 μM Y-27632 in aorta and BMA (Fig. 4, a and c), whereas responses of SMA were slightly attenuated (P < 0.05; Fig. 4b). Ro 31-8220 reduced contractile responses of SMA and abolished responses of BMA (P < 0.05; Fig. 4, b and c), but it had no effect in aorta (Fig. 4a). Furthermore, although U46619-induced contractile responses were also abolished in BMA during cotreatment with Y-27632 and Ro 31-8220 (P < 0.05; Fig. 4c), these inhibitors had a comparatively modest effect in aorta and SMA (P < 0.05; Fig. 4, a and b).

Discussion

This study has investigated the degree to which rho-kinase and PKC contribute to vascular contraction of large and small arteries within a vascular bed. The results reveal major segmental differences in the relative roles of rho-kinase and PKC in aorta, SMA, and BMA. First, in the larger vessels aorta and SMA, rho-kinase and PKC can each independently contribute to contractile responses to varying degrees. Second, in the small distal BMA, PKC seems to exclusively mediate these contractile responses with no apparent role for rho-kinase. Third, when used at a concentration of 10 μM, Y-27632 may exert nonspecific effects on PKC-mediated contractile responses.

Role of Rho-Kinase in Vascular Contraction. The role of rho-kinase in mediating contractile responses has been studied extensively (Bolz et al., 2000; Matrougui et al., 2001; Budzyn et al., 2006; Faraci et al., 2006). However, to the best of our knowledge, only one previous study has compared how the contribution of rho-kinase to contractile responses might vary between functionally distinct arteries within a vascular bed (Asano and Nomura, 2003). We found evidence for a role of rho-kinase in contractile responses to phenylephrine in both aorta and SMA, albeit to varying degrees. For example, responses of the aorta to phenylephrine were effectively abolished by rho-kinase inhibition, as we have demonstrated previously (Budzyn et al., 2004), as opposed to the relatively modest reduction in SMA. In stark contrast to the aorta, there was no effect of rho-kinase inhibition on phenylephrine-induced contractions of the small BMA. The rho-kinase inhibitor also modestly attenuated KCl-induced contractions in aorta and SMA, but it had no inhibitory effect in the smaller branch arteries. Thus, rho-kinase contributes to these contractile responses to markedly different degrees, according to the segment of the vascular bed. Interestingly, the modest attenuation of responses to KCl in the larger vessels, suggests a minor role for rho-kinase in the contractile response to this non-G protein-coupled receptor agonist, consistent with recent findings (Ratz et al., 2005).

Rho-kinase inhibition caused relatively weak attenuation of contractile responses to the thromboxane A2 mimetic U46619, in aorta and SMA. Thus, rho-kinase seems to play only a minor role in mediating contractile responses to this agent in these larger arteries. Previous studies examining the mechanisms of U46619-induced arterial contraction have reported variable findings with regard to the contribution of rho-kinase (Nobe and Paul, 2001; Tasaki et al., 2003; Wilson et al., 2005). Importantly, rho-kinase inhibition had no effect on contractile responses of BMA to all three contractile agents. Thus, in general, it seems that rho-kinase has a major role in mediating contractile responses of larger conductance vessels, whereas its contribution is greatly diminished in small resistance vessels (Asano and Nomura, 2003).

Role of PKC in Vascular Contraction. Recent studies using inhibitors with improved selectivity for PKC have yielded conflicting results regarding the physiological importance of that enzyme in the vasculature (Chrissobolis and Sobey, 2001; Shirao et al., 2002; McNair et al., 2004). Furthermore, little is known as to whether there are segmental differences in the role of PKC in vasoconstriction. We found evidence that PKC can contribute to contractile responses independently of rho-kinase, in each artery studied, albeit to varying degrees. For example, PKC inhibition with Ro 31-8220 had a more marked inhibitory effect on responses to phenylephrine and KCl in SMA than aorta, where rho-kinase inhibition was highly effective. Remarkably, Ro 31-8220 abolished responses of BMA to all contractile agents studied. However, contractile responses to U46619 were unaffected by PKC inhibition in aorta but significantly attenuated in SMA. Our finding that PKC had little apparent role in U46619-induced contractions of the aorta contrasts with two recent studies, suggesting that PKC does play a significant role in U46619-induced contractile responses in rat aorta and pulmonary vessels (Cogolludo et al., 2003; Tasaki et al., 2003). One reason for the different conclusions might be the different pharmacological inhibitors used. The precise roles of PKC in mediating U46619-induced contractile responses therefore still remain to be more definitively elucidated. Nevertheless, our findings clearly suggest that the importance of PKC in mediating vascular contraction is greater in resistance vessels such as second order branches of the SMA, relative to larger conductance vessels such as aorta where rho-kinase seems to dominate.

Generally, we found that combined rho-kinase and PKC inhibition resulted in near or complete abolition of all contractile responses in each artery studied, suggesting that a combination of these two kinases underlies the predominant mechanism(s) of contraction of perhaps most, if not all, blood vessels. However, although U46619-induced contractions of aorta or SMA were also attenuated by cotreatment with the two inhibitors, this effect was only modest. It is possible that the inhibitor-insensitive component of the response may actually be Ca2+-dependent, as suggested previously (Cogolludo et al., 2003; Tasaki et al., 2003) or might even involve alternative Ca2+-sensitizing mechanisms. We also acknowledge that we cannot exclude the possibility of an interaction between the rho-kinase and PKC pathways to induce contraction in the arteries studied. Some recent reports have provided evidence for such an interaction within the coronary and cerebral circulations (Kandabashi et al., 2003; Obara et al., 2005), yet whether the nature of this varies between vascular beds is still unclear.

PKC and Vascular Endothelium. We recently reported that in aorta, where the predominant endothelium-derived relaxing factor is NO, effectiveness of rho-kinase inhibition is much greater in the presence of endothelium and, more specifically, endothelial NO synthase activity (Budzyn et al., 2005). This observation is consistent with the concept that there are several levels of interaction between rho-kinase and endothelial NO (Takemoto et al., 2002; Budzyn et al., 2004; Wolfrum et al., 2004). In contrast to conductance arteries, in resistance arteries such as BMA, the predominant endothelium-derived relaxing agent is thought to be endothelium-derived hyperpolarizing factor (EDHF) rather than NO (Vanhoutte, 2004). We therefore tested whether, like rho-kinase and NO in aorta, PKC and EDHF can interact within the small BMA such that the effectiveness of Ro 31-8220 might be attenuated by endothelial removal. However, the dramatic effect of PKC inhibition on contractile responses of BMA was found to be unaffected by removal of endothelium, suggesting that EDHF is unlikely to modulate PKC-mediated contractile responses of this small artery.

Selectivity of Y-27632. Several of the present interpretations concerning the functional roles of rho-kinase rely on the selectivity of the pharmacological inhibitor Y-27632. Experimentally, Y-27632 has been used extensively to characterize both the physiological and pathophysiological roles of rho-kinase (Uehata et al., 1997; Chrissobolis and Sobey, 2001; Didion et al., 2005), and this agent is generally considered to be a highly selective rho-kinase inhibitor. We sought to test for potential nonspecific effects of Y-27632 on PKC by assessing its effect on contractile responses to the potent PKC activator PdB. Responses to PdB were abolished by Ro 31-8220, confirming that the PdB-induced contraction is entirely dependent on PKC activity. Interestingly, however, 10 μM but not 1 μM Y-27632 markedly inhibited responses to PdB. We interpret these data as indicating that at a concentration of 1 μM, the inhibitory effects of Y-27632 on vasoconstriction are probably due to selective inhibition of rho-kinase alone. However, at 10 μM (or higher concentrations), Y-27632 exerts nonspecific inhibitory effects on PKC-mediated vascular responses. Our results are in agreement with those of another study reporting that 10 μM Y-27632 inhibited phorbol ester-induced arterial contractions, in parallel with direct inhibition of PKCδ (Eto et al., 2001). Thus, past conclusions concerning the role of rho-kinase based on effects of 10 μMor higher of Y-27632 might warrant reevaluation.

Selectivity of Ro 31-8220. Unlike staurosporine and sphingosine, Ro 31-8220 is regarded as a relatively selective PKC inhibitor at the concentration used in the present study (5 μM), but it is not isoform-selective. It does, however, inhibit “conventional” PKC isoforms (α, β, and γ) more potently than “atypical” PKC isoforms (ϵ and ζ) (Davies et al., 2000). Eleven isoforms of PKC have been identified, and importantly, several of these are expressed in the VSM of the arteries studied (Salamanca and Khalil, 2005). Therefore, to elucidate precisely which PKC isoforms mediate the contractile effects in the arteries studied, further studies using isoform-specific inhibitors or antibodies might be warranted.

In summary, the present results provide strong functional evidence for rho-kinase and PKC having additive, yet differential roles in contractile responses of segmentally distinct arteries within a vascular bed. Furthermore, these roles may vary according to the contractile stimulus. We speculate that alterations to these signaling pathways during disease states may lead to diverse functional consequences depending on the vascular segment.

Footnotes

These studies were supported by Project Grant ID 208969 from the National Health and Medical Research Council of Australia (NHMRC). C.G.S. is a Senior Research Fellow and P.D.M. is a Principal Research Fellow of the NHMRC. K.B. is supported by a Melbourne University Research Scholarship.

References

Asano M and Nomura Y (2003) Comparison of inhibitory effects of Y-27632, a rho kinase inhibitor, in strips of small and large mesenteric arteries from spontaneously hypertensive and normotensive Wistar-Kyoto rats. Hypertens Res26:97-106.